Download In Silico Analysis: Annotations about Structural and Functional

Bangladesh J Microbiol, Volume 31, Number 1&2, June-December 2014, pp 53-58
Original Article
In Silico Analysis: Annotations about Structural and Functional Features of
DUF 2726 Family Member Proteins
SM Sabbir Alam, M Ruhul Amin, M Anwar Hossain*
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh
Domains of unknown functions (DUFs) are a big set of protein families within the Pfam database that
includes proteins of unknown function. In the absence of functional information, proteins are classified into
different families based on conserved amino acid sequences and are potentially functionally important. In
Pfam database, the numbers of families of DUFs are rapidly increasing and in current the fraction of DUF
families had increased to about twenty two percent of all protein families. In this study we targeted DUF2726
member proteins which are mainly present in different bacterial species of Gamma-proteobacteria and have
a particular domain organization. We analyzed the protein sequences of domain DUF2726 using different
computational tools and databases. We found that this domain contains a nuclear localization signal peptide,
which is conserved in Escherichia spp. and Shigella spp. It were also predicted that it has nucleic acid binding
properties. Analyzing protein-protein interactions functional partners associated with DUF 2726 were revealed.
Protein secondary structure, transmembrane helices structure were predicted. We have found that it has
gene neighbourhood and co-occurrences with protein RepA and RepB. RepA and RepB are functionally
associated with replication. RepA is a replication protein and RepB is a replication regulatory protein.
Presence of a nucleic acid binding properties, a nuclear localization signal (NLS) signalling peptide, and
possible interaction pattern with replication proteins, conjectures its possible role as a NLS like signalling
peptide.
Keywords: Domain of unknown function, DUF2726, Protein function prediction, NLS-like protein, Signalling
peptide.
Introduction
Pfam is a conserved protein family database and is widely used
to annotate and classify proteins1-2. Pfam has a database of curated
multiple sequence alignments for each of its all families. It also
contains structural and functional annotations, cross-database
links and citation references for each group. For each Pfam family
there have two multiple alignments: (1) the seed alignments which
contains a little number of representative members, and (2) the
full alignment containing all members in the database2-3.
The current version of Pfam contains about 14,831 protein
families. Compared with the previous version there have an
increase of 1,159 families and 16 new clans. Entire sequence
database (Pfam 23.0) contains about 5.3 million amino acid
sequences. And this number is increasing rapidly as growing
efficiency of genomic and metagenomic sequencing projects2-3.
DUFs, are a large set of families within the Pfam database that
includes only proteins of unknown function. The Pfam database
contains over 3,000 such families and the number of families of
DUFs is quickly increasing. It represents about 20% of known
protein families. Some DUF families are predicted to contain more
than one protein domains 1,4-6. Using different database
*Corresponding
information and alignment tools it was found that, 25% of the
DUF families are actually linked to some previously characterized
domain groups. Structural comparisons confirm these distant
relationships. Automated structure comparisons with manual
structure analysis it was found that these families can be classified
into known protein folds5,7.
To gain the ultimate goal of systems biology it is important to
know the function of all con-stituent parts of an organism. It was
found that even extensively studied organisms can have many
proteins unidentified or functionally unclassified. In yeast
Saccharomyces cerevisiae have around 1,000 proteins (~17% of
the genome) are still uncharacterized8.
In practical there have three ways to determine the function of an
uncharacterized domain – (1) identifying similarity to a known
function domain, either by sequence comparison or by structural
analysis, (2) using contextual information such as genomic context
to computationally identify function (e.g., STRING 9 and
PROLINKS10), and (3) using old-fashioned molecular biology or
biochemical techniques4. Various tools and databases are now
available to identify the relationships between DUFs and other
known families. Profile-HMM comparison tools, e.g., HHsearch11,
author:
M Anwar Hossain, Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh
Tel: +880 (02) 9661920-73, Ext 7735/7730; E-mail: [email protected]
Alam et. al
PRC12, SIMPRO 13 and SCOOP 14, have proved to be very helpful
in this perspect 4.
Secondary structure analysis
Secondary structure was analyzed using dompred22. DomPred is
a server designed to predict putative protein domains and their
boundaries for a given protein sequence23.
DUF 2726 family member proteins are mainly present on many
different bacterial species, especially of class Gammaproteobacteria and have a particular domain organization. This
domain is introduced in Pfam Release 24.0 in 20092 and is still
uncharacterized. A large portion of sequences of this domain
have came from organism Escherichia spp. and Shigella spp.
Finding relevant to our work we studied this group to predict its
function. We analyzed DUF2726 family member proteins in silico,
using different computational tools and databases to predict its
putative function.
Analysis for functional partners
Materials and Methods
STRING24 was used to determine functional partners of this
protein. The database STRING is a precomputed global resource
for the exploration and analysis of protein-protein associations.
It is used to retrieve and display the repeatedly occurring
neighbourhood of a gene. The repeated occurrence of genes in
each other’s neighbourhood on genomes has been shown to
indicate a functional association between the proteins they
encode25.
Data
Results
Domain information of DUF 2726 was found from Pfam database.
For analysis accession Q99QC5 was used as a sample sequence.
It is an uncharacterized protein of Shigella flexneri encoded in
gene yihA. Sequence data was downloaded from database
UniprotKB15.
Initial functional features were predicted by using PFP and SVM
prot. PFP result shows a different number of possible functional
classifications of protein (Table 1). PFP result showed that this
protein group may belong to many functional protein families
including, nucleotide binding properties. Further analysis was
done to verify and predict the correct functional properties of
this protein group. SVM prot shows similarity of this protein
with a number of protein families include: transmembrane metalbinding, nucleotide binding, hydrolases – acting on halide bonds,
DNA repair, copper-binding, calcium-binding, magnesium-binding
etc.
Function prediction of protein using PFP and SVM prot
Elementary functional prediction of protein was done by using
PFP16 and SVM prot17. PFP (Protein Function Prediction) is a
web-server for prediction of protein function developed by Kihara
Lab at Purdue University. It results most probable protein function
(GO terms) to given protein sequence based on its novel
algorithm16,18. SVM prot classifies proteins into functional family
from its primary sequence. SVM prot classification system is
trained from representative proteins of a number of functional
families and seed proteins of Pfam curated protein families17.
Presence of nuclear localization signal was analyzed using
predictNLS server. It was found that nuclear localization signal
was present in input sequence. Using Jalview it was found that
this nuclear localization sequence is conserved in Domain
organization of Shigella spp. and Escherichia coli. Conserved
nuclear localization sequence is ERRRRD. It is present in 176-181
a.a sequence. The conserved sequence was found from Jalview
shown in Figure 1.
Search for nuclear localization signal
PredictNLS server19 was used to determine the presence of nuclear
localization signal. NLSdb is a comprehensive source of
information regarding NLSs and proteins translocated into the
nucleus by signal sequences. Targeting signal recognition is a
key control point in the regulation of nuclear transport19. It is
therefore a used for identifying targeting signals in their sequence.
TMHMM result showed that input sequence may contain one
transmembrane helics. It has predicted that amino acid sequence
from 1-2 are present inside, 3-22 are present in transmembrane
region and 23-196 are present in outside region (Figure 2).
DomPred showed that DUF2724 contains more helix-residues
than strand and coil-residues. By using DomPred a probable
secondary structure of this domain group was predicted (Figure
3 and Figure 4).
Conservation analysis
Jalview20 was used to see conserveness of this localization signal
among domain of other sequences. It is a tool that is used to view
and edit multiple sequence alignments.
STRING was used to predict functional partners of this protein
(Table 2). Hypothetical functional partners are shown in Figure 5.
Among the functional partners of this protein, repA and repA2
are functionally associated with replication. RepA (285 aa) is a
replication protein and RepB (86 aa) is a replication regulatory
protein. RepA is of two types, repA2 and repA. repA2 is involved
in the determination of copy number in gene replication and RepA
play role as initiator protein for mRNA synthesis.
Transmembrane helices
TMHMM 16 was used to determine the presence of
transmembrane helices. TMHMM is a web based server
developed to predict TM helices and their topologies. It is a
method based on support vector machines (SVM) in a hierarchical
framework to predict TM helices first, followed by their topology
prediction21.
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In Silico Analysis of DUF 2726 Proteins
Table 1. Functional classification of protein from its primary sequences using PFP
Domain
name
DUF 2726
PFP result
Molecular function terms
Probability – Description
77% – Melanocortin receptor activity
67% – Melanocyte stimulating
hormone receptor activity
58% – Ion binding
57% – Binding
57% – Cation binding
56% – Transition metal ion binding
52% – Metal ion binding
50% – Protein binding
50% – Nucleotide binding
49% – Receptor binding
49% – Nucleic acid binding
49% – Adenyl nucleotide binding
46% – Peptide receptor activity
46% – Cytoskeletal protein binding
Biological process terms
Probability – Description
65% – Cellular metabolism
60% – Cellular physiological process
Cellular component terms
Probability – Description
75% – Voltage-gated sodium channel complex
60% – Cell
56% – Physiological process
56% – Cellular macromolecule metabolism
55% – Pyruvate family amino acid metabolism
54% – Cellular process
54% – Primary metabolism
53% – Macromolecule metabolism
53% – Metabolism
52% – Cellular biosynthesis
52% – Biopolymer metabolism
51% – Cellular protein metabolism
49% – Amino acid and derivative metabolism
49% – Nucleobase, nucleoside,
nucleotide and nucleic
acid metabolism
47% – Amine metabolism
46% – Organic acid metabolism
45% – Cellular catabolism
45% – Amine biosynthesis
44% – Transport
43% – Nitrogen compound biosynthesis
43% – Cell motility
42% DNA metabolism
41% – Locomotion
59% – Intracellular
59% – Membrane
56% – Cytoplasm
56% – Iintracellular organelle
55% – Plasma membrane
54% – Nucleus
53% – Intracellular membrane-bound organelle
53% – Exosome (RNase complex)
52% – Intracellular non-membrane-bound
organelle
52% – Cytoskeleton
43% – Integral to plasma membrane
45% – Peptide binding
43% – DNA binding
43% – Unfolded protein binding
41% – Purine nucleotide binding
40% – RNA binding
40% – Hormone binding
37% – Calcium ion binding
35% – Catalytic activity
34% – Actin binding
33% X-Pro dipeptidyl-peptidase
activity
33% – Peptide receptor activity, G-protein coupled
29% – Oxidoreductase activity
39%
35%
33%
28%
28%
28%
27%
20%
–
–
–
–
–
–
–
–
Integral to membrane
Non-membrane-bound organelle
Microtubule cytoskeleton
Membrane-bound organelle
Aminin-1
Aminin complex
Organelle
Microtubule
*Initial
functional features of query protein were predicted by using PFP and SVM prot. PFP result showed a different number of combinatorial possible
functional classifications of protein. It showed that this protein group may belong to many functional protein families including, nucleotide binding
properties.
Figure 1. Conserved NLS signal on domain of DUF2726. The conserved sequence of NLS signal (ERRRRD) was found to be present
in 176-181 aa in domain organization of Shigella spp. and Escherichia coli. Conserved NLS signal is shown in multiple sequence
alignment using Jalview.
55
Alam et. al
Figure 2. Results from TMHMM for DUF 2726. TMHMM was used to determine the amino acid positions that are inside/outside of
the transmembrane helices. It was found that amino acid positions from 23-196, that covers the NLS signal is located outside of the
transmembrane region.
Figure 3. Predicted secondary structure of protein Q99QC5.
Secondary structure was predicted using dompred. It showed that
DUF2724 contains more helix-residues than strand and coilresidues. The graph is derived by dompred from the N- and Ctermini positions from PSI-BLAST local alignments. Large values
indicate positions where sequence discontinuities occur, thus
locates putative domain boundaries. A putative domain is predicted
with the domain boundaries at residue positions of 100.
Figure 4. PSI-BLAST alignment profile. Predicted secondary
structure and PSI-BLAST alignment profile for Q99QC5
showing helix, strand and coil portions of different amino acids
and predicted confidence values.
Table 2. List of predicted functional partners found on STRING
* Hypothetical functional partners of YihA revealed by STRING. Functionally important protein- RepA and RepB are matched by gene neighborhood and
co-occurrence pattern.
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In Silico Analysis of DUF 2726 Proteins
Figure 5. Hypothetical functional partners of protein yihA explored by STRING. Protein network represents predicted functional
partners of protein yihA explored by STRING. Hypothetical functional partners are shown in 3D balls with associated interaction
pattern. Among the functional partners repA and repB are found functionally important and functionally associated with replication.
has gene neighbourhood and co-occurrences with protein repA
and repB. RepA and repB are functionally associated with
replication. RepA is a replication protein and RepB is a replication
regulatory protein. RepA2 is involved in the determination of
copy number in gene replication and RepA can play role as initiator
protein for mRNA synthesis. Presence of a nucleic acid binding
properties, a NLS signalling peptide, and possible interaction
pattern with other replication proteins, infers its possible role as
a NLS like signalling peptide associated with gene regulation.
Discussion
This study was targeted to analyze DUF2726 member proteins in
silico. DUF2726 family member proteins are mainly present on
different bacterial species of Gamma-proteobacteria and have a
unique domain organization. We had analyzed the protein
sequences of domain DUF2726 by utilizing different bioinformatics
tools and databases. We have found that this domain contains a
nuclear localization signal peptide, which is conserved in
Escherichia spp. and Shigella spp. In some previous studies it
was found that bacteria contain nuclear localization signal NLS
in their chromosome and/or plasmid and that NLS can work
efficiently upon infection of mammalian cells and has the
capability to transduce protein into mammalian cells26. It was
also found that some nucleoid associated proteins can play role
in gene regulation and horizontal gene transfer in enterobacteria27.
It was also found that some protein can have independent nuclear
and nucleoid localization functions28. In this study it was
predicted that it has nucleic acid binding properties. Analyzing
protein-protein interactions using STRING functional partners
associated with DUF2726 were explored. We have found that it
Conclusion
This study predicts that DUF2726 can act as a NLS like signalling
peptide. Data found from different tools or databases coincides
with the predicted function and showed significant evidence on
behalf of it. Further analysis in wet lab will verify this result.
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